In our earlier studies of regional cerebral blood flow using PET, we reported for the first time in humans that induced hypogonadism was associated with the elimination of the normal pattern of cortical activation in the dorsolateral prefrontal cortex as well as in the posterior inferior temporal cortices and the inferior parietal lobule;whereas both estradiol and progesterone replacement restored the normal pattern of cortical activation during a working memory task. We are pursuing our original findings that ovarian steroids modulate prefrontal cortical activity in women by augmenting older gold-standard imaging techniques (i.e., O15 PET), in which the technology is relatively stable over time and the activation task paradigms are kept relatively constant over the long-term course of these studies, with newer hypothesis-driven, cutting edge task paradigms and analytic approaches (i.e., fMRI). We employ several different activational paradigms during the neuroimaging procedures that will allow us to both vary the cognitive load of the task (and possibly the level of stress involved in performing the task) and test neural circuits more relevant to the phenomenology of affective disorders (i.e., reward system). For example, using fMRI and parametrically varied working memory load (i.e., N-back test), we are testing the possibility that change in hormonal state causes a shift in the dose-response curve between cognitive load and neurophysiological response. Additionally, using the N-back task with two functional imaging methods (PET with repeated scans and fMRI), we are determining the activation pattern for each woman at each hormonal phase to test for variance in their sites of activation. Finally, using new analytical approaches, we are testing the hypothesis that the hormonally-induced alteration in function includes changes in interregional functional connectivity either in the resting state or during activation. We have completed studies in over 35 asymptomatic women. Preliminary results from our O15 PET studies, demonstrate that estradiol significantly increases resting regional cerebral blood flow (rCBF) activity in regions of the default networks: medial prefrontal cortex (mPFC) and precuneous compared with the hypogonadal state. Progesterone significantly increases resting rCBF in the medial orbital frontal cortex (mOFC) and putamen compared with the hypogonadal state. Our findings that estradiol and progesterone regulate activities within mPFC and mOFC, respectively, have implications for understanding the potential impacts of both estradiol and progesterone on affective-adaptation and stress-responsivity. Preclinical studies have demonstrated that these brain areas are sites at which the effects of sex and sex-steroids modulate the effects of stress on brain function and behavior. Finally, recent data has documented the influence of genotype on cognitive performance and affective-adaptation. We now have identified for the first time in humans significant interactions between sex steroids and variations in specific genes regulated by ovarian steroids (i.e., catechol-o-methyl transferase and brain-derived neurotrophic factor) on measures of task-activated regional cerebral blood flow. Additionally, in a related project, we demonstrated changes in reward-related neurocircuitry across the normal menstrual cycle with increased activations during the expectancy of the reward during the follicular phase, when estradiol levels are high. These data demonstrate, for the first time in humans, that ovarian steroids modulate reward system function, with increased follicular phase activation of the orbitofrontal cortex and amygdala during reward anticipation and of the midbrain, striatum, and left ventrolateral prefrontal cortex (VLPFC) during reward delivery. We have further investigated these finding across the menstrual cycle by employing the reward paradigm in women who are participating in the GnRH agonist-induced hypogonadism study in which we examine the effects of estradiol and progesterone separately compared with a hypogonadal state. Preliminary findings demonstrate that both estradiol and progesterone modulate reward-related neurocircuitry. During the anticipation of reward, estradiol increased activity in the amygdala and orbitofrontal cortex compared with the hypogonadal state (consistent with our previous data across the menstrual cycle), and progesterone increased activity within the right inferior frontal gyrus compared with both estradiol and hypogonadism. These data suggest a substrate in which changes in gonadal steroids could modulate affective state, and are consistent with preclinical studies that document the modulatory effects of ovarian steroids on reward-related behaviors and brain regions, as well as a growing literature demonstrating the importance of the reward system in depression. We are awaiting completion of the analyses of possible diagnostic differences (i.e., PMD versus controls) in ovarian steroid-related alterations of reward neurocircuitry. In our previous studies of the effects of gonadal steroids on stress responsivity, we identified that progesterone rather than estradiol has a greater impact on HPA axis activation in humans, unlike in rodents, but the response to progesterone appears to differ in women with PMD and controls. We have conducted dexamethasone-corticotrophin releasing hormone (dex/CRH) testing in women with PMD and controls in both menstrual cycle and Lupron studies, as this measure is believed to be particularly sensitive to differences in vasopressin. We have completed a study of 43 women employing Dex/CRH studies. Data confirm a progesterone-related enhancement of HPA axis (see Annual Report for project MH002865-05). There has been a widespread presumption in clinical neuroscience that ovarian steroids regulate 5HT transport function leading to sex and sex steroid-related differences in affective adaptation. We are over halfway through the recruitment of our investigations of the effects of ovarian steroids on serotonin transporter function. In these studies, we employ the 5HT transporter ligand DASB during both naturalistic and experimentally-induced conditions to examine claims that ovarian steroids are important physiological regulators of the 5HT transporter. These data will be the first to characterize the effects of sex steroids on5HT transporter function in the CNS, and will either confirm or seriously question the physiologic relevance of the widely embraced assumption that special relationship exists between ovarian steroids and 5HT function.